The zymogen granule is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and is a classic model for studying secretory granule function. Our long term goal is to develop a comprehensive architectural model for zymogen granule membrane (ZGM) proteins that would direct new hypotheses for subsequent functional studies. Our initial proteomics analysis focused on identification of proteins from purified ZGM (Mol Cell Proteomics 5, 306-312, 2006). In the current study, a new global topology analysis of ZGM proteins is described that applies isotope enrichment methods to a protease protection protocol. Our results showed that tryptic peptides of ZGM proteins were separated into two distinct clusters according to their iTRAQ ratios for proteinase K treated vs control ZGs. The low iTRAQ ratio cluster included cytoplasm-orientated membrane and membrane-associated proteins including Myosin V, VAMPs, Syntaxins and all the Rab proteins. The second cluster having unchanged ratios included predominantly lumenal proteins. Because quantification is at the peptide level, this technique is also capable of mapping both cytoplasm- and lumen-orientated domains from the same transmembrane protein. In order to more accurately assign the topology, we developed a statistical mixture model to provide probabilities for identified peptides to be cytoplasmic or luminal based on their iTRAQ ratios. By implementing this approach to global topology analysis of ZGM proteins, we report here an experimentally constrained, comprehensive topology model of identified ZG membrane proteins. This model contributes to a firm foundation for developing a higher order architecture model of the ZGM and for future functional studies of individual ZGM proteins.